With recent technical advances, multiple important signaling pathways that may be the causes of human malignancy have continuously been discovered and dissected. The vast majority of these signaling pathways involve reversible protein phosphorylation, and the information on the location and dynamics of phosphorylation provides important mechanisms on how the signaling networks function and interact. While translational research gradually shifts from lab models to clinical samples, with the ultimate goal of identifying cancer biomarkers, a simple and reliable phosphorylation assay method is still missing for routine detection of phosphorylation in complex and typically heterogeneous clinical samples. Through this NIH SBIR Phase I study, we will further develop a novel strategy for phosphorylation assay, termed pIMAGO (phospho-imaging) that has recently been introduced by us, into commercial products for simple, routine phosphorylation assays. This novel design takes advantage of not only the quantum size properties of the soluble nanoparticles, but also of the multi-functionalized nature of the molecule, allowing for highly selective, sensitive and simple qualitative and quantitative assessment of protein phosphorylation without the use of either radioactive isotopes or expensive phosphospecific antibodies. Due to its size and unique properties, it also offers the capability for multiplexed detection of phosphorylation and total protein amount simultaneously. We propose to optimize the technology for on-membrane phosphoprotein detection in routine biomedical research. In addition, we will develop a novel fluorescence-based pIMAGO reagent for direct detection and multiplexed experiments to add another dimension for biomedical research and development. PUBLIC HEALTH RELEVANCE: Protein phosphorylation relates to the onset and development of many cancer types and a highly efficient technology for phosphorylation analysis is critical for cancer research. This NIH SBIR will support an effort to develop an innovative technology into commercial products that equip researchers with powerful tools and new directions to combat the devastating disease.